Plants are known to produce a variety of chemical compounds, either constitutively or inducibly, to protect themselves against environmental stresses, wounding, or microbial invasion.
Of the plant antimicrobial proteins that have been characterized to date, a large proportion share common characteristics. They are generally small (<10 kDa), highly basic proteins and often contain an even number of cysteine residues (typically 4, 6 or 8). These cysteines all participate in intramolecular disulfide bonds and provide the protein with structural and thermodynamic stability (Broekaert et al. (1997)). Based on amino acid sequence identities, primarily with reference to the number and spacing of the cysteine residues, a number of distinct families have been defined. They include the plant defensins (Broekaert et al., 1995, 1997; Lay et al., 2003a), thionins (Bohlmann, 1994), lipid transfer proteins (Kader, 1996, 1997), hevein (Broekaert et al., 1992) and knottin-type proteins (Cammue et al., 1992), as well as antimicrobial proteins from Macadamia integrifolia (Marcus et al., 1997; McManus et al., 1999) and Impatiens balsamina (Tailor et al., 1997; Patel et al., 1998) (Table 1). All these antimicrobial proteins appear to exert their activities at the level of the plasma membrane of the target microorganisms, although it is likely that the different protein families act via different mechanisms (Broekaert et al., 1997). The cyclotides are a new family of small, cysteine-rich plant peptides that are common in members of the Rubiaceae and Violaceae families (reviewed in Craik et al., 1999, 2004; Craik, 2001). These unusual cyclic peptides (Table 1) have been ascribed various biological activities including antibacterial (Tam, et al., 1999), anti-HIV (Gustafson et al., 1994) and insecticidal (Jennings et al., 2001) properties.
TABLE 1Small, cysteine-rich antimicrobial proteins in plants.Rep-No. ofPeptideresentativeaminofamilymemberacidsConsensus sequencePlant defensinsRs-AFP251 α/β-Thionin (8-Cys type)α-Purothionin45 Lipid transfer proteinAce-AMP193 Hevein- typeAc-AMP230 Knottin- typeMj-AMP136 MacadamiaMiAMP176 ImpatientsIb-AMP120 CyclotideKalata B129
The size of the mature protein and spacing of cysteine residues for representative members of plant antimicrobial proteins is shown in Table 1. The numbers in the consensus sequence represent the number of amino acids between the highly conserved cysteine residues in the representative member but other members of the family may vary slightly in the inter-cysteine lengths. The disulfide connectivities are given by connecting lines. The cyclic backbone of the cyclotides is depicted by the broken line (from Lay and Anderson, 2005).
Defensins
The term “defensin” has previously been used in the art to describe a diverse family of molecules that are produced by many different species and which function in innate defense against pathogens including bacteria, fungi, yeast and viruses.
Plant Defensins
Plant defensins (also termed γ-thionins) are small (˜5 kDa, 45 to 54 amino acids), basic proteins with eight cysteine residues that form four strictly conserved disulfide bonds with a CysI-CysVIII, CysII-CysIV, CysIII-CysVI and CysV-CysVII configuration. As well as these four strictly conserved disulfide bonds, some plant defensins have an additional disulfide bond (Lay et al., 2003a, 2003b; Janssen et al., 2003).
The name “plant defensin” was coined in 1995 by Terras and colleagues who isolated two antifungal proteins from radish seeds (Rs-AFP1 and Rs-AFP2) and noted that at a primary and three-dimensional structural level these proteins were distinct from the plant α-/β-thionins but shared some structural similarities to insect and mammalian defensins (Terras et al., 1995; Broekaert et al., 1995).
Plant defensins exhibit clear, although relatively limited, sequence conservation. Strictly conserved are the eight cysteine residues and a glycine at position 34 (numbering relative to Rs-AFP2). In most of the sequences, a serine at position 8, an aromatic residue at position 11, a glycine at position 13 and a glutamic acid at position 29 are also conserved (Lay et al., 2003a; Lay and Anderson, 2005).
The three-dimensional solution structures of the first plant defensins were elucidated in 1993 by Bruix and colleagues for γ1-P and γ1-H. Since that time, the structures of other seed-derived and two flower-derived (NaD1 and PhD1) defensins have been determined (Lay et al., 2003b; Janssen et al., 2003). All these defensins elaborate a motif known as the cysteine-stabilized αβ (CSαβ) fold and share highly superimposable three-dimensional structures that comprise a well-defined α-helix and a triple-stranded antiparallel β-sheet. These elements are organized in a βαββ arrangement and are reinforced by four disulfide bridges.
The CSαβ motif is also displayed by insect defensins and scorpion toxins. In comparing the amino acid sequences of the structurally characterized plant defensins, insect defensins and scorpion toxins, it is apparent that the CSαβ scaffold is highly permissive to size and compositional differences.
The plant defensin/γ-thionin structure contrasts to that which is adopted by the α-and β-thionins. The α-and β-thionins form compact, amphipathic, L-shaped molecules where the long vertical arm of the L is composed of two α-helices, and the short arm is formed by two antiparallel β-strands and the last (˜10) C-terminal residues. These proteins are also stabilized by three or four disulfide bonds (Bohlmann and Apel, 1991).
Plant defensins have a widespread distribution throughout the plant kingdom and are likely to be present in most, if not all, plants. Most plant defensins have been isolated from seeds where they are abundant and have been characterized at the molecular, biochemical and structural levels (Broekaert et al., 1995; Thomma et al., 2003; Lay and Anderson, 2005). Defensins have also been identified in other tissues including leaves, pods, tubers, fruit, roots, bark and floral tissues (Lay and Anderson, 2005).
An amino acid sequence alignment of several defensins that have been identified, either as purified protein or deduced from cDNAs, has been published by Lay and Anderson (2005). Other plant defensins have been disclosed in U.S. Pat. No. 6,911,577, International Patent Publication No. WO 00/11196 and International Patent Publication No. WO 00/68405, the entire contents of which are incorporated herein by reference.
Mammalian Defensins
The mammalian defensins form three distinct structural subfamilies known as the α-, β-and θ-defensins. In contrast to the plant defensins, all three subfamilies contain only six cysteine residues which differ with respect to their size, the placement and connectivity of their cysteines, the nature of their precursors and their sites of expression (Selsted et al., 1993; Hancock and Lehrer, 1998; Tang et al., 1999a, b; Lehrer and Ganz, 2002). All subfamilies have an implicated role in innate host immunity and more recently, have been linked with adaptive immunity as immunostimulating agents (Tang et al., 1999b; Lehrer and Ganz, 2002). It was in the context of their defense role that the name “defensin” was originally coined (Ganz et al., 1985; Selsted et al., 1985).
The α-defensins (also known as classical defensins) are 29-35 amino acids in length and their six cysteine residues form three disulfide bonds with a CysI-CysVI, CysII-CysIV and CysIII-CysV configuration (Table 2).
In contrast to the α-defensins, the β-defensins are larger (36-42 amino acids in size) and have a different cysteine pairing (CysI-CysV, CysII-CysIV and CysIII-CysVI) and spacing (Tang and Selsted, 1993). They are also produced as preprodefensins. However, their prodomains are much shorter. Analogous to the α-defensins, the synthesis of β-defensins can be constitutive or can be induced following injury or exposure to bacteria, parasitic protozoa, bacterial lipopolysaccharides, and also in response to humoral mediators (i.e. cytokines) (Diamond et al., 1996; Russell et al., 1996; Tarver et al., 1998).
The size of the mature protein and spacing of cysteine residues for representative members of defensin and defensin-like proteins from insects and mammals is shown in Table 2. The numbers in the consensus sequence represent the number of amino acids between the highly conserved cysteine residues in the representative member, but other members of the family may vary slightly in the inter-cysteine lengths. The disulfide connectivities are given by connecting lines. The cyclic backbone of the mammalian theta-defensins is depicted by the broken line.
TABLE 2Representative members of defensin and defensin-like proteins from insects and mammalsRepresentativeNo. ofPeptide familymemberamino acidsConsensus sequenceReferenceInsect defensin-likeDrosomycin44Lamberty et al., 2001 Insect defensinInsect defensin A40Cornet et al., 1995 Mammalian α-defensinHNP-434Harwig et al., 1992 Mammalian β-defensinHBD-136Bensch et al., 1995 Mammalian θ-defensinRTD-118Tang et al., 199a, b Trabi et al., 2001Insect Defensins
A large number of defensin and defensin-like proteins have been identified in insects. These proteins are produced in the fat body (equivalent of the mammali an liver) from which they are subsequently released into the hemolymph (Lamberty et al., 1999). Most insect defensins have three disulfide bonds. However, a number of related proteins, namely drosomycin from Drosophila melanogaster, have four disulfides (Fehlbaum et al., 1994; Landon et al., 1997) (Table 2).
The three-dimensional structures of several insect defensins have been solved (e.g. Hanzawa et al., 1990; Bonmatin et al., 1992; Comet et al., 1995; Lamberty et al., 2001; Da Silva et al., 2003). Their global fold, as typified by insect defensin A, features an α-helix, a double-stranded antiparallel β-sheet and a long N-terminal loop. These elements of secondary structure are stabilized by three disulfide bonds that are arranged in a CysI-CysIV, CysII-CysV and CysIII-CysVI configuration (Bonmatin et al., 1992; Cornet et al., 1995).
Two Classes of Plant Defensins
Plant defensins can be divided into two major classes according to the structure of the precursor proteins predicted from cDNA clones (Lay et al., 2003a) (FIG. 1). In the first and largest class, the precursor protein is composed of an endoplasmic reticulum (ER) signal sequence and a mature defensin domain. These proteins enter the secretory pathway and have no obvious signals for post-translational modification or subcellular targeting (FIG. 1A).
The second class of defensins are produced as larger precursors with C-terminal prodomains or propeptides (CTPPs) of about 33 amino acids (FIG. 1B). Class II defensins have been identified in solanaceous species where they are expressed constitutively in floral tissues (Lay et al., 2003a; Gu et al., 1992; Milligan et al., 1995; Brandstadter et al., 1996) and fruit (Aluru et al., 1999) and in salt stressed leaves (Komori et al., 1997; Yamada et al., 1997). The CTPP of the solanaceous defensins from Nicotiana alata (NaD1) and Petunia hybrida (PhD1 and PhD2) is removed proteolytically during maturation (Lay et al., 2003a).
The CTPPs on the solanaceous defensins have an unusually high content of acidic and hydrophobic amino acids. Interestingly, at neutral pH, the negative charge of the CTPP counter-balances the positive charge of the defensin domain (Lay and Anderson, 2005).
Biological Activity of Plant Defensins
Some biological activities have been attributed to plant defensins including growth inhibitory effects on fungi (Broekaert et al., 1997; Lay et al., 2003a; Osborn et al., 1995; Terras et al., 1993), and Gram-positive and Gram-negative bacteria (Segura et al., 1998; Moreno et al., 1994; Zhang and Lewis, 1997). Some defensins are also effective inhibitors of digestive enzymes such as α-amylases (Zhang et al., 1997; Bloch et al., 1991) and serine proteinases (Wijaya et al., 2000; Melo et al., 2002), two functions consistent with a role in protection against insect herbivory. This is supported by the observation that bacterially expressed mung bean defensin, VrCRP, is lethal to the bruchid Callosobruchus chinensis when incorporated into an artificial diet at 0.2% (w/w) (Chen et al., 2002). Some defensins also inhibit protein translation (Mendez et al., 1990; Colilla et al., 1990; Mendez et al., 1996) or bind to ion channels (Kushmerick et al., 1998). A defensin from Arabidopsis halleri also confers zinc tolerance, suggesting a role in stress adaptation (Mirouze et al., 2006). More recently, a sunflower defensin was shown to induce cell death in Orobanche parasite plants (de Zélicourt et al., 2007).
Antifungal Activity
The best characterized activity of some but not all plant defensins is their ability to inhibit, with varying potencies, a large number of fungal species (for examples, see Broekaert et al., 1997; Lay et al., 2003a; Osborn et al., 1995). Rs-AFP2, for example, inhibits the growth of Phoma betae at 1 μg/mL, but is ineffective against Sclerotinia sclerotiorum at 100 μg/mL (Terras et al., 1992). Based on their effects on the growth and morphology of the fungus, Fusarium culmorum, two groups of defensins can be distinguished. The “morphogenic” plant defensins cause reduced hyphal elongation with a concomitant increase in hyphal branching, whereas the “non-morphogenic” plant defensins reduce the rate of hyphal elongation, but do not induce marked morphological distortions (Osborn et al., 1995).
More recently, the pea defensin Psd1 has been shown to be taken up intracellularly and enter the nuclei of Neurospora crassa where it interacts with a nuclear cyclin-like protein involved in cell cycle control (Lobo et al., 2007). For MsDef1, a defensin from alfalfa, two mitogen-activated protein (MAP) kinase signaling cascades have a major role in regulating MsDef1 activity on Fusarium graminearum (Ramamoorthy et al., 2007).
Permeabilization of fungal membranes has also been reported for some plant defensins (Lay and Anderson, 2005). For example, NaD1 is a plant defensin isolated from floral tissue of Nicotiana alata. The amino acid and coding sequences of NaD1 are disclosed in International Patent Publication No. WO 02/063011, the entire contents of which are incorporated by reference herein. NaD1 was tested in vitro for antifungal activity against the filamentous fungi Fusarium oxysporum f. sp. vasinfectum (Fov), Verticillium dahliae, Thielaviopsis basicola, Aspergillus nidulans and Leptosphaeria maculans. At 1 μM, NaD1 retarded the growth of Fov and L. maculans by 50% while V. dahliae, T. basicola, and A. nidulans were all inhibited by approximately 65%. At 5 μM NaD1, the growth of all five species was inhibited by more than 80%. These five fungal species are all members of the ascomycete phylum and are distributed among three classes in the subphylum pezizomycotiria. These fungi are agronomically important fungal pathogens. All filamentous fungi tested thus far are sensitive to inhibition by NaD1 (van der Weerden et al., 2008).
The importance of the four disulfide bonds in NaD1 was investigated by reducing and alkylating the cysteine residues. Reduced and alkylated NaD1 (NaD1R&A) was completely inactive in the growth inhibitory assays with Fov, even at a concentration ten-fold higher than the IC50 for NaD1 (van der Weerden et al., 2008).
Prior Work with Antimicrobial Peptides and Tumour Cells
Use of Small Cysteine-Rich/Cationic Antimicrobial Peptides in the Treatment of Human Disease
There is an increasing body of literature implicating human α-and β-defensins in various aspects of cancer, tumourigenesis, angiogenesis and invasion. The use of mammalian defensins has also been proposed for the treatment of viral and fungal infections and as an alternative or adjunct to antibiotic treatment of bacterial infections. However, their cytotoxicity towards mammalian cells remains a significant barrier. Moss et al (U.S. Pat. No. 7,511,015) have shown that modification of the defensin peptide through ribosylation or ADP-ribosylation of arginine residues modifies the toxicity of the peptide and enhances its antimicrobial properties.
The review by Mader and Hoskin (2006) describes the use of cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment. It should be noted however that a review by Pelegrini and Franco (2005) incorrectly describes α-/β-thionins from mistletoe, which are anticancer molecules, as γ-thionins (another name for plant defensins). The person skilled in the art would understand that such prior art does not relate to plant defensins (γ-thionins) but instead to the structurally and functionally distinct α-/β-thionins.
Reports of Plant Defensins with Antiproliferative Activity on Human Cancer Cells
Since 2004, some isolated reports have suggested that plant defensin(-like) proteins could also display in vitro antiproliferative activity against various human tumour cell lines (with differing potencies) (see, for example, Wong and Ng (2005), Ngai and Ng (2005), Ma et al. (2009) and Lin et al. (2009)). These proteins have largely been isolated from leguminous plants (e.g. beans). The assignment of these proteins lathe plant defensin class was based on their estimated molecular mass (˜5 kDa) and in some cases, on limited N-terminal amino acid similarities to known defensin sequences. However, the proteins as disclosed in these references lack the strictly conserved cysteine residues and cysteine spacings that define defensins. In addition, the proteins disclosed in such references are not Class II defensins, nor are they from the family Solanaceae.
A review of the literature indicates that the Capsicum chinese defensin (CcD1), also referred to as Cc-gth, was the only other Class II defensin of the Solanaceae family that has been previously implicated as having the potential to inhibit the viability of mammalian cells (Anaya-Lopez et al., 2006). It is reported that the transfection of an expression construct encoding a full-length sequence for CcD1 into the bovine endothelial cell line BE-E6E7 resulted in conditioned media that exhibited anti-proliferative effects on the human transformed cell line HeLa. There are a number of major flaws in the experimental design and interpretation of these data that make it impossible for the person skilled in the art to draw a valid conclusion from the described studies as to whether CcD1 exhibits anti-proliferative activity. These include: (i) although mRNA for CcD1 was suggested in the transfected cells, no evidence was provided to demonstrate that the CcD1 protein was actually expressed in the conditioned media, (ii) the use of the full-length open-reading frame of CcD1 rather, than the mature coding domain would require the processing of the expressed precursor by removal of the CTPP domain to produce an “active” defensin—this was not demonstrated, (iii) the process of transfection can result in changes to a cell and the control for the transfection experiment was not adequate in that untransfected cells were used rather than the correct control of vector alone transfected cells, (iv) the use of conditioned media rather than purified CcD1 protein could influence the experimental readout as components of the media or other secreted molecules from the transfected cells may themselves, or in combination with CcD1, have anti-proliferative activity, (v) the expression levels of CcD1 mRNA in the various transfected endothelial cell populations (Anaya-Lopez et al., 2006, FIG. 2) do not correlate with the proposed anti-proliferative activity of the CcD1 transfected cell conditioned media (Anaya-Lopez et al., 2006, FIG. 4) as there is no statistically significant difference between the observed anti-proliferative responses mediated by the different conditioned media samples. It should also be noted that these deficiencies in the experimental design and interpretation were expressly acknowledged in an independently published paper by the same authors in 2008 (Loeza-Angeles et al., 2008). Based on these observations, it would be impossible for the person skilled in the art to interpret from Anaya-Lopez et al. (2006) that CcD1 has any anti-proliferative activity against mammalian cells.
The inventors have previously disclosed in International Patent Publication No. WO 02/063011 certain novel defensins and their use in inducing resistance in plants or parts of plants to pathogen infestation. The entire contents of WO 02/063011 are incorporated herein by reference.
As a result of follow up studies into plant defensins, the inventors have also previously disclosed in International Patent Publication No. WO 2011/160174 that Class II defensins from the Solanaceae plant family have potent cytotoxic properties. These significant findings described a novel and important way in which proliferative diseases may be prevented and treated. The entire contents of WO 2011/160174 are incorporated herein by reference.
As a result of yet further studies into plant defensins, it has been determined that a previously undisclosed Class II defensin from the Solanaceae plant family has potent cytotoxic properties that are surprisingly coupled with a very high IC50 and hence a very high degree of specificity for tumour cells, as opposed to normal, healthy cells. Accordingly, these findings provide for vastly improved compositions and methods for the prevention and treatment of proliferative diseases such as cancer, as well as associated systems and kits. Such compositions, methods, systems and kits provide a hitherto unseen degree of specific targeting against tumour cells versus normal, healthy cells, and therefore minimize side effects. Such compositions also allow for much higher safe doses of treatment, thereby facilitating a much improved degree of efficacy in treatment.